Molecular basis of fusB-mediated resistance to fusidic acid in Staphylococcus aureus

The primary mechanism of fusidic acid resistance in clinical strains of Staphylococcus aureus involves acquisition of the fusB determinant. The genetic elements(s) responsible are incompletely defined, and the mechanism of resistance is unknown. Here we report the cloning, sequencing and overexpression of a single gene (fusB) from plasmid pUB101 capable of conferring resistance to fusidic acid in S. aureus. The fusB gene is located on a transposon-like element and encodes a small (25 kDa), cytoplasmic protein for which homologues exist in a number of clinically important and environmental Gram-positive bacterial species. Bioinformatic analysis of regions immediately upstream of fusB suggested that expression of resistance is regulated by translational attenuation, which was confirmed through use of reporter fusions. FusB was overexpressed in Escherichia coli as a polyhistidine-tagged fusion product, and the purified protein shown to protect an in vitro staphylococcal translation system from inhibition by fusidic acid in a specific and dose-dependent fashion. Purified FusB bound staphylococcal EF-G, the target of fusidic acid. The protein provided no protection from inhibition by fusidic acid when added to an in vitro E. coli translation system, consistent both with the observed failure of FusB to bind E. coli EF-G, and its inability to confer resistance in E. coli.     

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem-iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem-binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter-like beta-sandwich fold with a large hydrophobic haem-binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co-ordinated solely by Tyr(166). The phenol groups of Tyr(166) and Tyr(170) form an H-bond that may function in regulating haem binding and release. An analysis of IsdA structure-sequence alignments indicate that conservation of Tyr(166) is a predictor of haem binding by NEAT domains.     

Molecular design of multifunctional antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA)

Antibacterial activity of a series of alkyl gallates (3,4,5-trihydroxybenzoates) against Gram-positive bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains was evaluated. Gram-positive bacteria are all susceptible to alkyl gallates. Dodecyl gallate was the most effective against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate was bactericidal against this MRSA strain at any growth stage. This activity was observed even in the chloramphenicol-treated cells, but the rate of decrease of cell number was slower than that in the exponentially growing cells. The bactericidal activity of medium-chain alkyl gallates was noted in combination with their ability to disrupt the native membrane-associated function nonspecifically as surface-active agents (surfactants) and to inhibit the respiratory electron transport. Subsequently, the same series of alkyl protocatechuates (3,4-dihydroxybenzoates) were studied and the results obtained are similar to those found for alkyl gallates. The length of the alkyl chain is not a major contributor but is related to the activity.     

Characterization of hemin antibacterial action on Staphylococcus aureus

Abstract The mechanism of inactivation of Staphylococcas aureus cells by hemin is described. Protection experiments by sulfhydryl reagents such as cysteine, mercaptoethanol, glutathione or thioglycolate in their reduced form prevent S. aureus bacteria from inactivation by hemin (1.5 × 10⁻⁵ M). The treatment of bacteria by hemin in the presence of one of those reagents (1 × 10⁻² M) showed that the growth rate and viability of the culture remained unchaged. On the other hand sulfhydryl reagents did not prevent the binding of hemin to the bacteria. When cysteine or glutathione were introduced to a culture after exposure to hemin it could neither reverse the damage done to the cells nor shorten the time of the culture's recovery. Another type of protection was obtained by addition of serum albumin which prevented hemin molecules from binding to the bacterial envelopes. Furthermore, when albumin was introduced after the bacteria were treated by hemin it prevented further damage to the survivors and thus shortened the time required for recovery. None of the singlet oxygen quenchers or hydroxyl radical scavengers could protect the bacteria from hemin inactivation. The mechanism by which hemin affects S. aureus is assumed to be by oxidizing a major system within the cell.     

Promising antibacterial agents against multidrug resistant Staphylococcus aureus

Rapid emergence of multidrug resistant Staphylococcus aureus infections has created a critical health menace universally. Resistance to all the available chemotherapeutics has been on rise which led to WHO to stratify Staphylococcus aureus as high tier priorty II pathogen. Hence, discovery and development of new antibacterial agents with new mode of action is crucial to address the multidrug resistant Staphylococcus aureus infections. The egressing understanding of new antibacterials on their biological target provides opportunities for new therapeutic agents. This review underlines on various aspects of drug design, structure activity relationships (SARs) and mechanism of action of various new antibacterial agents and also covers the recent reports on new antibacterial agents with potent activity against multidrug resistant Staphylococcus aureus. This review provides attention on in vitro and in vivo pharmacological activities of new antibacterial agents in the point of view of drug discovery and development.     

Structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14

Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9–sgRNA–dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.     

Development of novel antibacterial agents against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health because of its resistance to multiple antibiotics most commonly used to treat infection. In this study, we report the unique ability of the cyclooxygenase-2 (COX-2) inhibitor celecoxib to kill Staphylococcus aureus and MRSA with modest potency. We hypothesize that the anti-Staphylococcus activity of celecoxib could be pharmacologically exploited to develop novel anti-MRSA agents with a distinct mechanism. Examination of an in-house, celecoxib-based focused compound library in conjunction with structural modifications led to the identification of compound 46 as the lead agent with high antibacterial potency against a panel of Staphylococcus pathogens and different strains of MRSA. Moreover, this killing effect is bacteria-specific, as human cancer cells are resistant to 46. In addition, a single intraperitoneal administration of compound 46 at 30 mg/kg improved the survival of MRSA-infected C57BL/6 mice. In light of its high potency in eradicating MRSA in vitro and its in vivo activity, compound 46 and its analogues warrant continued preclinical development as a potential therapeutic intervention against MRSA.     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.     

Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase

Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug-an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.     

Molecular basis for bre5 cofactor recognition by the ubp3 deubiquitylating enzyme

Yeast Ubp3 and its co-factor Bre5 form a deubiquitylation complex to regulate protein transport between the endoplasmic reticulum and Golgi compartments of the cell. A novel N-terminal domain of the Ubp3 catalytic subunit forms a complex with the NTF2-like domain of the Bre5 regulatory subunit. Here, we report the X-ray crystal structure of an Ubp3-Bre5 complex and show that it forms a symmetric hetero-tetrameric complex in which the Bre5 NTF2-like domain dimer interacts with two L-shaped beta-strand-turn-alpha-helix motifs of Ubp3. The Ubp3 N-terminal domain binds within a hydrophobic cavity on the surface of the Bre5 NTF2-like domain subunit with conserved residues within both proteins interacting predominantly through antiparallel beta-sheet hydrogen bonds and van der Waals contacts. Structure-based mutagenesis and functional studies confirm the significance of the observed interactions for Ubp3-Bre5 association in vitro and Ubp3 function in vivo. Comparison of the structure to other protein complexes with NTF2-like domains shows that the Ubp3-Bre5 interface is novel. Together, these studies provide new insights into Ubp3 recognition by Bre5 and into protein recognition by NTF2-like domains.     

Independent recognition of Staphylococcus aureus by two receptors for phagocytosis in Drosophila

Integrin βν, one of two β subunits of Drosophila integrin, acts as a receptor in the phagocytosis of apoptotic cells. We here examined the involvement of this receptor in defense against infection by Staphylococcus aureus. Flies lacking integrin βν died earlier than control flies upon a septic but not oral infection with this bacterium. A loss of integrin βν reduced the phagocytosis of S. aureus and increased bacterial growth in flies. In contrast, the level of mRNA of an antimicrobial peptide produced upon infection was unchanged in integrin βν-lacking flies. The simultaneous loss of integrin βν and Draper, another receptor involved in the phagocytosis of S. aureus, brought about a further decrease in the level of phagocytosis and accelerated death of flies compared with the loss of either receptor alone. A strain of S. aureus lacking lipoteichoic acid, a cell wall component serving as a ligand for Draper, was susceptible to integrin βν-mediated phagocytosis. In contrast, a S. aureus mutant strain that produces small amounts of peptidoglycan was less efficiently phagocytosed by larval hemocytes, and a loss of integrin βν in hemocytes reduced a difference in the susceptibility to phagocytosis between parental and mutant strains. Furthermore, a series of experiments revealed the binding of integrin βν to peptidoglycan of S. aureus. Taken together, these results suggested that Draper and integrin βν cooperate in the phagocytic elimination of S. aureus by recognizing distinct cell wall components, and that this dual recognition system is necessary for the host organism to survive infection.     

耐甲氧西林金黄色葡萄球菌的分子流行病学研究

了解我院患者耐甲氧西林金黄色葡萄球菌（MRSA）的分子流行病学特点,为临床抗感染治疗提供依据。收集2007年1月~2008年9月我院分离的耐甲氧西林金黄色葡萄球菌共54株,采用PCR进行SCCmec基因分型、葡萄球菌A蛋白（SPA）分型,并检测杀白细胞毒素（PVL）基因,同时应用脉冲场凝胶电泳（PFGE）进行同源性分析。54株MRSA菌株SCCmec基因分型为SCCmecⅡ型17株,SCCmecⅢ型33株,SCCmecⅣ型2株,SCCmecⅤ型2株;SPA基因分型将28株归属为t030,9株为t002,8株为t037,5株为t570,2株为t437,t163和t796各1株;PVL毒素检测只有2株SCCmecⅣ型菌株阳性;PFGE证实院内MRSA感染主要为2种克隆株传播,同时还有其他型别出现。本院MRSA流行传播的SCCmec基因型主要以Ⅲ型占优势,同时发现有携带PVL毒素的CA-MRSA分离株流行,应引起密切关注。     

Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.     

Accumulation of porphyrins and pyrrole pigments by Staphylococcus aureus ssp. anaerobius and its aerobic mutant

Abstract The anaerobic, Gram-positive coccus Staphylococcus aureus ssp. anaerobius and its aerobic mutant MVF-SR, when kept under anaerobic conditions, excreted coproporphyrin (mainly type III) into the medium and enriched uroporphyrin (mainly type I) within the cells.
The rate of porphyrin synthesis stayed practically unaltered when the growth medium was supplemented with 50 μ g/ml 5-aminolevulinic acid (ALA), but was significantly enhanced upon supplementation with hemin (0.5 μ g/ml). When hemin and ALA were given simultaneously, a more than two-fold increase in porphyrin production compared to normal growth medium was observed. These observations indicate a stimulation of porphyrin synthesis in S. aureus by hemin.
An as yet unidentified violet pigment with an intense red-violet fluorescence under UV light ( λ = 366 nm) was found to be present in considerable amounts in cells of S. aureus ssp. anaerobius , whereas the supernatant medium of aerobically grown cells of the mutant MVF-SR contained an equally unidentified blue, non-fluorescing pigment.     

Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin

The structure of the 28 kDa complex of the first two RNA binding domains (RBDs) of nucleolin (RBD12) with an RNA stem-loop that includes the nucleolin recognition element UCCCGA in the loop was determined by NMR spectroscopy. The structure of nucleolin RBD12 with the nucleolin recognition element (NRE) reveals that the two RBDs bind on opposite sides of the RNA loop, forming a molecular clamp that brings the 5' and 3' ends of the recognition sequence close together and stabilizing the stem-loop. The specific interactions observed in the structure explain the sequence specificity for the NRE sequence. Binding studies of mutant proteins and analysis of conserved residues support the proposed interactions. The mode of interaction of the protein with the RNA and the location of the putative NRE sites suggest that nucleolin may function as an RNA chaperone to prevent improper folding of the nascent pre-rRNA.